Tuberculosis is a leading cause of death from infection worldwide. Investigations of interactions between Mycobacterium tuberculosis (Mtb) and human macrophages are fundamental for understanding tuberculosis pathogenesis. Immunostimulatory DNA sequences and their synthetic oligonucleotide analogs (CpG-ODN) have been shown to exert antibacterial effects in vitro and in vivo, and we discovered that CpG-ODN activate anti-mycobacterial activity in human monoeyte-derived macrophages (hMDM). The goals of this K08 project are to more fully characterize the mechanism(s) and magnitude of this CpG-ODN effect. Experiments will determine whether anti-mycobacterial responses are activated thorugh TLR9, the only receptor known to be activated by CpG-ODN. While murine macrophages can be activated by interferon (IFN)-gamma to kill intracellular mycobacteria in vitro, we and others have found that IFN-gamma, paradoxically potentiates intracellular growth of the pathogen. The mechanisms whereby human macrophages restrict Mtb remain uncertain, and new knowledge may be gained through delineating the mechanisms acitvated by CpG-ODN. We will determine if this response is broadly applicable to different Mtb strains, and if there are differences between the activity of hMDM and human alveolar macrophages (the initial host cell infected by Mtb in vivo). We will also test whether macrophage-like cell lines (THP-1) can be used to model the CpG-ODN response, since these cells would facilitate molecular studies of Toll-dependent signaling. Clues to the mechanism of this CpG-ODN effect will be sought in functional studies ofmacrophage activation and signaling, and using microarrays to interrogate the transcriptome of stimulated cells broadly. Finally, we will compare the effects of stimulation through different Toll receptors with a panel of ligands to look for similarities or differences compared with CpG-ODN, and to identify potential synergies. These studies will provide new basic knowledge of the innate anti-mycobacterial functions of human macrophages, and they will provide a rational basis for the discovery of new immunotherapeutic strategies for tuberculosis.